title: [Establishment of a high efficient human coagulation factor VIII eukaryotic expression system using lentiviral vector].
abstract: OBJECTIVE
  To establish a high efficient human coagulation factor VIII (FVIII) eukaryotic stable expression system using lentiviral vector, and determine its biosafety.
  
  
  METHODS
  Lentiviral transfer plasmid carrying human B-domain-deleted FVIII(BDDhFVIII)-IRES-GFP(BDDhFVIII/pXZ9)or IRES-GFP(pXZ9) was constructed. Lentivirus particles were produced by transiently co-transfected 3-plasmids into 293FT cells and further concentrated via ultracentrifugation. CHO cells were infected, 72h later, the FVIII antigen (FVIII:Ag) concentration in the medium was examined by ELISA, the activity was detected via one stage coagulation,and the transcription of FVIII in the infected CHO cells was determined by RT-PCR.Virus infection ability in the medium and the gag gene in CHO cells were determined to evaluate the model's biosafety.
  
  
  RESULTS
  Lentiviral transfer plasmid BDDhFVIII-IRES-GFP(BDDhFVIII/pXZ9)carrying human B-domain-deleted FVIII or IRES-GFP (pXZ9) was successfully constructed, and high titer lentiviruses has been prepared. The lentivirus could infect CHO cells efficiently, after an additional 72 h, the FVIII:Ag concentration had up to (1724.9±283.7) mU/ml, the FVIII:C level increased to (10.58±1.55)%, and transcripts of BDDhFVIII mRNA could be measured by RT-PCR. Neither the gag gene nor the virus in the supernatant was detected.
  
  
  CONCLUSIONS
  Lentivirus-mediated human coagulation factor VIII could be expressed efficiently in CHO cells. The system couldn't produce offspring virus, proving a good biosafety.
authors.affiliation: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors.forename: Xu-guang
authors.initials: XG
authors.lastname: Song
authors.forename: Jiang
authors.initials: J
authors.lastname: Cao
authors.forename: Ling-yu
authors.initials: LY
authors.lastname: Zeng
authors.forename: Huan-xin
authors.initials: HX
authors.lastname: Zhang
authors.forename: Hai
authors.initials: H
authors.lastname: Cheng
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Chong
authors.initials: C
authors.lastname: Chen
authors.forename: Kai-lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D014162:Transfection
chemical_list: D005169:Factor VIII